Unrelated Donor Cord Blood Transplantation in Children: Lessons Learned Over 3 Decades

Abstract Four decades ago, Broxmeyer et al. demonstrated that umbilical cord blood (CB) contained hematopoietic stem cells (HSC) and hypothesized that CB could be used as a source of donor HSC for rescue of myeloablated bone marrow. In 1988, Gluckman et al. reported the first successful matched sibling cord blood transplant (CBT) in a child with Fanconi Anemia. In 1991, Rubinstein et al. established an unrelated donor CB bank, and in 1993, the first unrelated CBT used a unit from this bank. Since that time, >40 000 CBTs have been performed worldwide. Early outcomes of CBT were mixed and demonstrated the importance of cell dose from the CB donor. We hypothesized that improvements in CB banking and transplantation favorably impacted outcomes of CBT today and performed a retrospective study combining data from Eurocord and Duke University in 4834 children transplanted with a single unrelated CB unit (CBU) from 1993 to 2019. Changes in standard transplant outcomes (overall survival [OS], disease free survival [DFS], acute and chronic graft-versus-host disease [GvHD], treatment related mortality [TRM], and relapse) over 3 time periods (1: <2005; 2: 2005 to <2010; and 3: >2010 to 2019) were studied. Increased cell dose and degree of HLA matching were observed over time. OS, times to engraftment, and DFS improved over time. The incidence of TRM and GvHD decreased while the incidence of relapse remained unchanged. Relative contributions of cell dose and HLA matching to transplant outcomes were also assessed and showed that HLA matching was more important than cell dose in this pediatric cohort.


Introduction
Forty years ago, Hal Broxmeyer discovered that umbilical cord blood (CB) contained hematopoietic stem and progenitor cells (HSCs), 1 leading to the use of CB as an alternative donor for hematopoietic stem cell transplantation (HSCT). CB is rapidly available, enables permissive HLA-matching, low rates of graft versus host disease (GvHD), and enhanced graft versus leukemia (GvL) effects. Historically, CB was associated with slower hematopoietic engraftment and immune reconstitution, higher infectious complications and transplant-related mortality (TRM). [2][3][4] Early trials demonstrated that cell dose (CD) was rate limiting for engraftment, 2,3 The fact that CB is the only banked source of HSCs led to regulation as a commercial product resulting in increased costs compared to nonregulated HSC sources.
Outcomes of CBT have improved since the early days of implementation. [2][3][4][5][6][7][8] Publicly banked CBUs are larger 9 resulting in shorter times to engraftment and reduced TRM. The use of dCBT and the emergence of ex-vivo expansion technology have decreased time to engraftment, days of hospitalization, and TRM. 10 In experienced centers, outcomes of CBT are equivalent or superior to outcomes of HSCT using other donor sources. 7,[11][12][13] Despite these benefits, the use of CBT has decreased worldwide. Much of this decline is related to the use of haplo-identical related donors enable by the development of innovative strategies to prevent GvHD. 14 In light of these trends, we investigated outcomes after CBT in a large international cohort of children treated over the past 3 decades. We asked key questions about the interactions between HLA matching and cell dose (CD), the role of anti-thymocyte globulin (ATG), and the role of total body irradiation (TBI) in patients with leukemia.

Methods
This multicenter retrospective study was conducted through a joint collaboration with Eurocord/European Blood and Marrow Transplant Group (EBMT) and the Pediatric Transplant and Cellular Therapy Program at Duke University. The aim of the study was to describe trends in outcomes of unrelated CBT in children over 28 years.

Study Design
Eligible patients were children (<18 years of age) who received an unrelated single unit CBT, as their first allogeneic HSCT between 1993 and 2019, for hematological malignancies and non-malignant diseases. Patients who received other products or a second allogeneic transplant were excluded. Data related to patient demographics (excluding race/ethnicity, which is not permitted to be collected in France), CBU characteristics, and transplant outcomes were analyzed. The Institutional Review Boards of the Eurocord scientific committee and Duke University approved this study. All patients or their parents or legal guardians gave informed consent for treatment, data entry, and analysis in accordance with the Declaration of Helsinki.

Endpoints and Definitions
The primary endpoint was overall survival (OS), defined as the probability of being alive regardless of disease status. Secondary outcomes included the cumulative incidence (CI) of neutrophil recovery, acute and chronic GvHD (aGvHD and cGvHD), GvHD, Relapse Free, Survival (GFRF), and TRM, defined as any death not caused by relapse or persistent malignancy. Disease-free survival (DFS), defined as survival while in continuous complete remission (CR), and relapse were evaluated in patients with malignant diseases. Surviving patients who did not experience any of these events were censored at the time of last follow-up. Neutrophil engraftment was defined as the achievement of a sustained absolute neutrophil count greater than 0.5 × 10 9 /L for 3 consecutive days. Graft failure was defined as failure to achieve neutrophil engraftment, loss of donor engraftment, or autologous recovery within day +100 after CBT. The diagnosis and grading of acute and chronic GvHD were assigned by the transplant center using standard criteria. The conditioning regimen was defined as myeloablative (MAC) or reduced intensity (RIC) based on EBMT and CIBMTR criteria. CD was calculated as the total number of pre-cryopreservation and infused total nucleated cells (TNC)/kg and CD34 cells/kg, respectively. Human leukocyte antigen (HLA) matching between the recipient and the UCB donor was determined conventionally considering antigen-level HLA typing (low resolution) for Class I-A and -B loci and allele-level (high resolution) for Class II-DRB1 locus. A subset of patients had HLA allelic typing.

Statistical Analysis
Calendar time was divided into 3 periods for analysis of transplant outcomes: P1-1990 to <2005, P2-2005 to <2010, and P3-2010 to 2019. Overall survival (OS) was analyzed using the Kaplan-Meier method. TRM, relapse, acute, and chronic GvHD, and time to neutrophil recovery were analyzed using the CI method for competing risks. Competing events were relapsed (in the analysis of TRM) or death (in the analysis of relapse, neutrophil recovery, and acute and chronic GvHD). Unadjusted comparisons of these outcomes across calendar periods were accomplished using the log-rank test (OS) or Gray's test (all other outcomes). Multivariable models were fit describing the association between calendar period and each of the outcomes after adjustment for use of ATG, HLA mismatch, age at transplant, pre-transplant serum positivity for cytomegalovirus (CMV), malignant versus non-malignant indication for transplant, pre-cryopreservation TNC dose/kg of patient weight (TNC/kg), MAC vs. RIC conditioning regimen, and use of TBI. All covariates were included in every model regardless of statistical significance to allow evaluation of the association between each covariate and the transplant outcomes after adjustment for the other factors. Cox proportional hazards regression was used to model overall survival and the Fine and Gray model was used for the other outcomes that have competing events. A total of 33.9% of participants were missing data on at least one of the model covariates, with the highest missingness rate for an individual covariate being 16.7%. Missingness in covariates was related to calendar period (9.6%, 29.5%, and 55.8% missing at least one covariate in the early (P1), middle (P2), and later (P3) periods, respectively) and was modestly associated with older age at transplant and malignant disease. Prior to fitting any models, all missing covariate data were imputed (20 datasets) 15 using the fully conditional specification method. Imputations of continuous variables were restricted to be within the range of the observed data. Analysis of OS (no missing outcomes), neutrophil engraftment, and relapse (missing in <1% of participants) were based on models fit to the available data after covariate imputation. Analysis of TRM, aGvHD, and cGvHD was based on inverse probability weighted models (IPW) 16 as these outcomes were missing in 10-25% of participants. Weights were estimated (after covariate imputation) using logistic regression as the inverse of the predicted probability of having an observed outcome based on the covariates used in the analysis model described above. Results of models fit to each of the imputed datasets were combined using Rubin's rules 17 and summary hazard ratios (HR) and 95% CIs were reported. All analyses were conducted using SAS v9.4 (Cary, NC) with PROC MI, MIANALYZE, PHREG, and GENMOD. The TNC dose/kg increased significantly over time with a median precryopreservation dose/kg from 6.66 × 10 7 in P1 to 9.29 × 10 7 in P3 (P = <.0001) ( Table 2). The same trends were observed with the infused TNC and CD34 cell doses. We were not able to include infused TNC or CD34 cell doses in the multivariate analyses because of missing data (for infused TNC) and collinearity concerns for CD34. Both the precryopreservation and infused TNC/kg (median 8.07 and 6.17 × 10 7 ) and infused CD34 cell doses/kg (median 2.30 × 10 5 ) were high in this pediatric cohort.

Patient, Disease, and Transplant Characteristics
HLA matching was scored at low resolution for Class I HLA-A, -B and high resolution for Class II (HLA-DRB1) for  S4). There was also an increase in DRB1 matching from P1 to P3 76.4% increasing to 82.7%. HLA by high resolution typing was available for 1466 patient/CB pairs at 8 loci and the same trend to more HLA compatibility was observed with a doubling of patients receiving grafts with 0 or 1 HLA mismatches (P = <.0001) ( Table 2, Supplementary Fig. S1).

Causes of Death
The causes of death by year of transplant are shown in Supplementary Table S1. The main causes of death were relapse/disease progression (n = 628, 30.1%) and TRM including GvHD (n = 1220, 58.4%), secondary malignancy (n = 12), other or missing (n = 229). GvHD was responsible for TRM in 19.6% of patients overall and was relatively stable  Table S2). In patients with malignancies, rates of death from relapse remained stable while TRM decreased over time (Fig. 3), but TRM remained the leading cause of death in patients with malignancies over time. Earlier year of transplantation (P = <.001), increasing HLA mismatch (P = <.002), positive CMV serology pretransplant (P = .001), precryo TNCC (P = .18), and older age at transplant (P = .011) were associated with increased risk of death (Table 3). There was also a trend (P = .66) toward increased mortality with use of ATG (Supplementary Table S3).

Notable Outcomes in Patients with ID
Multivariable analysis of death in patients with ID showed a higher risk with more HLA mismatches and older age. CI of neutrophil engraftment ranged between 79.2% and 83.7% at day 60 and was not influenced by year of transplant (P = .378). The CI of aGvHD decreased from 33% (95% CI: 23.7, 42.6) in P1 to 18.3% (95% CI: 13.9, 23.1) in P3 (P = .0048) and was impacted by year of transplant and use of ATG in multivariable analysis. The CI of cGvHD fell from 20% (12, 29.4) to 11.5% (7.2, 16.9) (P = .1816). In multivariable analysis there was a trend for a lower risk of cGvHD in younger age, use of ATG, closer HLA matching, and more recent time of transplant.

Other Key Observations
We investigated the relative contributions of HLA matching and CD in this large patient cohort. Over time, both administered CD and degree of HLA match increased. In addition, in these pediatric patients, CDs exceeded established minimal thresholds. We extracted the relative role of these 2 parameters from the multivariable analyses for patients with malignant and patients with non-malignant conditions for all-cause mortality, TRM, relapse (in patients with malignancies), neutrophil engraftment, and acute and chronic GvHD (Supplementary Fig. S5). Overall, closer HLA matching was more likely than CD to favorably influence outcomes in this group of pediatric patients receiving an adequate CD from a single CBU.
ATG was administered in the majority of transplants over time (92% of patients transplanted in P1, 77.9% in P2, and 86.4% in P3). We saw evidence that suggested the impact of ATG on TRM and all-cause mortality depended on calendar period. Specifically, ATG was associated with a trend towards reduced risk of TRM but with an increasingly larger effect over calendar time (HR = 0.6, HR = 0.35, and HR = 0.31 in P1, P2, and P3; P = .066). However, ATG use was associated with an increased risk of all-cause mortality in P1 that  Fig. S6). The role of TBI in patients with leukemias in preventing relapse, increasing DFS, and contributing to TRM was also investigated. While there was no change in the incidence of relapse over time, the incidence of relapse in patients with ALL was lower when TBI was administered as part of the conditioning regimen (Supplementary Table S4).

Discussion
We report results of CBT in a large cohort of children receiving a single unit, unrelated CBT over the past 3 decades. Several important observations are highlighted. First, OS improved over time from 41.8% before 2005 to 60% after 2010 (P < .0001). This is likely to be attributed to use of more closely matched and higher CD containing CB grafts. Patient related factors, including selection and treatment in earlier stages of disease, type of conditioning, selected use of posttransplant maintenance therapy, and more effective prevention and treatment modalities GvHD and infection prevention and newer and more effective treatment modalities further improved outcomes. Despite higher CDs and improved HLA matching there is still a relationship between the two in that HLA matching influenced all the outcomes including survival, TRM, engraftment, and GvHD whereas CD was important for survival and engraftment. All in all, in this population of children receiving adequate to higher doses of cells from a single CBU, closer HLA matching was more significant in favorably influencing outcomes compared to CD.
Identifying a CBU with an adequate CD, above the TNC threshold of 2.5 × 10 7 /kg, for children is not a problem due to their lower body weight. Overall, the pre-cryopreservation CB CD increased from 6.6 to 8.57 × 10 7 /kg over time. In multivariable analysis for engraftment, we found that a higher CD was associated with higher rates of engraftment compared to an inferior CD (P < .001) while increasing the CD above the median of TNC 6.9 × 10 7 /kg did not further improve engraftment. Thus, once this CD is reached, HLA matching should be prioritized l,2,18,19 Also, increasing CD beyond the median did not correlate with lower TRM or further improve OS. In earlier periods, CB unit selection was based on HLA-A and -B antigen typing and DR-B1 allelic typing. The number of patients with HLA-A, -B, -C, and DRB1 allele typing increased overtime representing 50% of the patients after 2010. Using low resolution typing, the number of patients with 6/6 and 5/6 HLA matching increased significantly over time to 74% after 2010 (P = .0001). The same trend was observed when considering patients who had high resolution typing with 79.6% of patients receiving a 5/8-8/8 matched CBU. DRB1 matching was prioritized over class I matching with 81.4% patients receiving a DRB1 matched CBU, 52% HLA-A matched, 44% HLA-B matched, and 38.4% HLA-C matched.
The complex interaction between CD and HLA parity and their impact on CBT outcomes was studied by several groups that demonstrated that increasing TNC dose resulted in higher engraftment rates and lower TRM. [20][21][22] In 2007, Eapen and colleagues reported, in a pediatric cohort of 785 patients with leukemia who received unrelated bone marrow transplant or CBT, a better 5-year LFS after transplants with a 0/6 low resolution HLA mismatched CB compared to bone marrow, but no difference in engraftment of CB with 1-2/6 mismatches and bone marrow transplantation. 21 The following year Kurtzberg et al. in a prospective study including 191 unrelated CBT in children with hematologic malignancies, showed improved OS with units having <2/6 mismatches, and higher TNC doses. 20 Eapen and colleagues (2014) studied a cohort of 1568 patients (mainly children) and reported higher TRM with increasing HLA high resolution mismatches (9% for 0/8; 26% for 1-2/8; and 34 % for 3/8). 22 CBTs have the advantage of lower incidence of acute and chronic GvHD. 23,24 However, the impact of HLA disparity remains unclear, and many reports failed to predict the occurrence of aGvHD based on low resolution HLA disparity, 2,3,25,26 although Rubinstein et al. showed lower frequency of severe (grades III-IV) aGvHD in patients with 6/6 HLA antigen matches (P = .008). In a retrospective registry-based review, the incidence of grade II-IV aGvHD was 39% after single CBT, and risk of aGvHD was found to increase with the degree of allelic HLA mismatches. 22 Acute GvHD was also reported to correlate with an increased risk of cGvHD. 27,28 In our cohort, the incidence of aGvHD was 33.5% similar to other published results. 22 The correlation we found in multivariable analysis between ATG administration and risk of aGvHD was expected. However, we found no difference in the incidence of aGvHD based on HLA disparity (low-or high-resolution typing) unlike other studies that showed higher risk of developing aGvHD with mismatches at high resolution typing. 20,22 Our findings are likely different due to the preferential selection of the CBUs with relatively high TNC doses which might be able to abrogate the effect of increasing levels of HLA mismatches. Importantly, in our cohort, aGvHD did not correlate with a high incidence of cGvHD, and only 17.5% of the children developed cGvHD at 5 years. Low resolution HLA disparity was the only factor associated with the increasing risk of cGvHD.
Positive pretransplant CMV serology appears to be an important prognostic factor in this population despite improvements in detection and treatment of CMV reactivation. Patients with a positive CMV serology before transplant had a higher risk of death, lower engraftment but a lower risk of GvHD in multivariable analysis. The association of a positive CMV serology with adverse outcomes has previously been described after HSCT from alternative donors. The combination of CB donor, which is by definition CMV negative, and the patient who could be either CMV seropositive or negative presents an opportunity to further study CMV infection and immune reconstitution after transplant.

Conclusion
In conclusion, studying a large cohort of children transplanted with a single CBU for standard indications for HSCT over the past 3 decades, we found improved engraftment and survival and decreased TRM over time. Current CBT outcomes favorably compare with other donor sources utilized in patients lacking a matched related donor. [29][30][31] Public CB bank inventories have expanded and represent prequalified, high quality, "off-the-shelf" donors that are readily available for shipment and use. When selecting a CBU for a pediatric patient, prioritize HLA-matching over CD when an adequate dose is available from their best available CBU.

Author Contributions
J.K.: conception and design, administrative support, provision of study material and patients, collection and/or assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript. T.D.J.: collection and/or assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript. P.M.K.: conception and design, administrative support, provision of study material and patients, collection and/or assembly of data, data analysis and interpretation, collection and/or assembly of data, manuscript writing, final approval of manuscript. H.R.El.A.: collection and/or assembly of data, manuscript writing, final approval of manuscript. F.V.: collection and/or assembly of data, manuscript writing, final approval of manuscript. G.M.S.: manuscript writing, final approval of manuscript. B.C.: manuscript writing, final approval of manuscript. V.R.: data analysis and interpretation, manuscript writing, final approval of manuscript. A.R.: provision of study materials and patients, collection and/or assembly of data, data analysis and interpretation manuscript writing, final approval of manuscript. E.G.: conception and design, provision of study material and patients, collection and/or assembly of data, data analysis and interpretation, collection and/or assembly of data, manuscript writing, final approval of manuscript.

Data Availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.